Bacteriophage replicase

Sequencing.—With the elucidation of the primary and secondary structure of the replicase gene, the complete 3569-nucleotide-long sequence of the RNA of bacteriophage MS2 is now known.153 This is the first organism for which theentire nucleic acid structure has been elucidated, and Fiers and his group richly deserve their bouquet. 

Some E. coli bacteriophages, including f2, MS2, R17, and Qj8, as well as some eukaryotic viruses (including influenza and Sindbis viruses, the latter associated with a form of encephalitis) have RNA genomes. The single-stranded RNA chromosomes of these viruses, which also function as mRNAs for the synthesis of viral proteins, are replicated in the host cell by an RNA-dependent RNA polymerase (RNA replicase). All RNA viruses—with the exception of retroviruses—must encode a protein with RNA-dependent RNA polymerase activity because the host cells do not possess this enzyme. 

The RNA replicase of most RNA bacteriophages has a molecular weight of -210,000 and consists of four subunits. 

RNA-dependent RNA polymerases, such as the replicases of RNA bacteriophages, are template-specific for the viral RNA. 

Figure 29-17 Partial sequence and secondary structure model of RNA of bacteriophage MS2. The initiation and termination codons for each of the three genes (A protein, coat protein, and replicase) are enclosed in boxes as is the second stop signal that is in-frame for the A protein gene but out-of-frame for the coat protein gene. The entire coat protein gene is shown but less them one-third of the entire sequence is given. From W. Fiers and associates.499-501...

Fig. I. The single-stranded RNA of the bacteriophage Q(3 is reproduced with the assistance of an enzyme, called Q(3 replicase, which consists of four subunits (black dots). The enzyme recognizes the matrix specifically and during synthesis, it moves from the 3 to the 5 end of the template strand. The replica formed (-) is complementary to the template (+). The 3 and 5 ends are symmetrically related in such a way that both plus- and minus-strands have similar 3 ends both are recognized by the replicase, and the minus-strand thus acts as a template for the formation of a plus-strand. Internal folding of the two strands prevents the formation of a plus-minus double helix.

The RNA genomes of single-stranded RNA bacterial viruses, such as Q/3, MS2, R17, and f2, are themselves mRNAs. Bacteriophage Q/3 codes for a polypeptide that combines with three host proteins to form an RNA-depen-dent RNA polymerase (replicase). The three host proteins are ribosomal protein SI and two elongation factors for protein synthesis EF-Tu and EF-Ts (see table 28.5). The Q/3 replicase functions exclusively with the Q/3 RNA plus strand template. It first makes a complementary RNA transcript (minus strand) and ultimately uses the minus strand as... 

The first successful attempts to study RNA evolution in vitro were carried out in the late sixties by Sol Spiegelman9 and his group at Columbia University (Spiegel-man, 1971). They made use of an RNA replicase isolated from Escherichia coli cells infected by the RNA bacteriophage QP and prepared a medium for replication by adding the four ribonucleoside triphosphates (GTP, ATP, CTP, and UTP) in a suitable buffer solution. QP RNA, when transferred into this medium, instantaneously started to replicate. Evolutionary experiments were carried out by means of the serial transfer technique (Figure 4). Materials consumed in RNA replication... 

A search for a specific inhibitor of the action of RNA replicase (RNA dependent RNA polymerase) another point at which viral replication could be inhibited independently of host cell metabolism, led to the screening of KXX) compounds against bacteriophage Q/S RNA replicase with E. coli as host cell. Thirty active compounds were obtained of which (LXIV) was the most active. This inhibited phage multiplication in E. coli by 99 per cent compared with control at 10 pg/ml whereas host cell growth was only inhibited by 50 per cent [243]. These authors hopefully conclude thus, further studies may produce a universal anti-RNA virus drug without side effects . 

A., Volckaert, G., and Ysebaert, M. (1976) Complete nucleotide-sequence of bacteriophage MS2-RNA - primary and secondary structure of replicase gene. Nature, 260, 500-507. 

It was suggested that poly(c) tracts mi t play a role in the replication of those viruses which contain it (28, 29) In particular, they speculated that such a poly(c) tract mi t function as a replicase-binding site, or a part of one, on the basis of two observations. First, Rosenberg at al. (39) reported that a partially purified EMC viral replicase preparation will synthesise poly(G) from a poly(c) template, but will not copy other synthetic primers. Second, the replicase of bacteriophage which has subunits of similar size to those of the EMCV replicase (39) can also specifically use poly(C) as a template for poly(G) synthesis. However, the significance of this specificity is unclear, since Q3... 

Infection of animal cells with a picomavirus results in the formation of an RNA dependent RNA polymerase (replicase) which is apparently responsible for the biosynthesis of the viral RNA genome. The discovery of the picomavirus replicase (l-3) and similar enzymatic activities in E, coli infected with RNA bacteriophages (4-6) were made about fifteen years ago. Today, there is detailed knowledge of the structure and properties of the replicases of bacteriophage Qg (7i 8) and f2 (9)j but only partial infoimation on the picomavirus replicase (10-14) This rather slow progress is due mainly to the difficulties encountered in the isolation of a stable ENA dependent replicase from a eukaryotic cell-vims system. 

A comparison of the bacterial and animal systems with respect to development of an enzyme isolation method indicates the disadvantage of the latter. There is a much smaller amount of replicase in animal cells infected with a picornavims than in E. coli infected with bacteriophage Qg. Thus, about 180-300 units of Qg replicase are found in 1 g of infected E. coli (15)> only 1-2 units of EMC vims replicase in 1 g of infected B cells (13) ... 

The RNA replication complex of a picornavims is bound to smooth cytoplasmic membranes (10, 16). In order to obtain a soluble RNA dependent activity it is necessary to dissociate the enzyme from the membranes by means which adversely affect the activity of the enzyme. It is also more laborious and costly to grow and infect large quantities of animal cells than to carry out a large scale infection of E. coli with an RNA bacteriophage. So far, because of these limitations, the isolation of a picornavims replicase was carried out with very dilute solutions of enzyme-protein, a... 

In E. coli, translational control of the three cistrons of Q, f2, and related bacteriophage RNAs (Fig. 12) accounts for the synthesis of coat protein replicase A protein in the approximate ratio of 20 5 1. These quantitative differ-... 

Fig. 8. The technique of serial transfer experiments (Spiegelman 1971). RNA, in particular RNA of the bacteriophage Q, grows in a medium which contains the enzyme Q replicase as well as the four nucleoside triphosphates ATP, UTP, GTP and CTP in excess. After a time At a sample is taken out of the test tube. Part of it is analysed, part of it is transferred into a new medium. The procedure is repeated after time intervals of At.

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